The human genome has the potential to encode ~350 different ubiquitin ligase enzymes that are based on a cullin-RING catalytic core, making the cullin-RING ligases (CRLs) one of the largest known superfamilies of enzymes. In keeping with the large number of CRLs, members of this superfamily have been implicated in regulating many aspects of cell and organismal biology - ranging from nutrient sensing to control of circadian rhythms. Based on their extraordinary diversity and profound impact on biology, it is important that we understand how these enzymes work, how they are controlled, and how they might be manipulated for the benefit of human health. The activity of CRLs is regulated by the ubiquitin-like peptide, NeddS, which is covalently attached to the cullin subunit. All cullins are subjected to cycles of attachment ('neddylation') and removal ('deneddylation') of NeddS. Cullins modified by NeddS are assembled into active CRLs, whereas unmodified cullins can become sequestered into inactive complexes with a sequestration factor named CAND1. In this application, I propose three Specific Aims to evaluate novel hypotheses for how attachment of NeddS stimulates the ubiquitin ligase activity of CRLs (Aim 1), and how the cycles of cullin neddylation and deneddylation are coupled to the assembly and activity of CRLs (Aim 2). Finally, I propose to discriminate between different models for how cullins are emancipated from CAND1 so that they can nucleate assembly of an intact, functional CRL (Aim 3). These Aims will be pursued by applying a combination of biochemical reconstitution, fluorescence-based measurement of protein interactions (both dynamically and at equilibrium), and analysis of the assembly and modification state of mutant CRL subunits in vivo. Defining mechanisms of action and regulation for these enzymes will enable us to understand in greater depth their contribution to human regulatory biology as well their role in sustaining diseases (of which several are known, including cancers and inflammatory diseases) whose progression relies on the function of one or more CRL. Moreover, detailed knowledge of the mechanism of action and regulation of this class of enzymes may assist in the development of small molecule-based therapeutic strategies for modulating the activity of CRLs to alleviate symptoms of disease or stem biochemical processes that underlie disease.